1. Field of the Invention
This invention relates to the preparation of oligosaccharides, using polymer supported methodology. By this method, which offers anomeric control, oligosaccharides are produced rapidly and in good yield in comparison with known methodologies.
2. Description of Related Art
Oligosaccharides may be elaborated into glycopeptides and glycolipids which have important utility in the fields of medicine, biotechnology, and related technologies. Oligosaccharides have been synthesized by solution methodologies for many years as reviewed, for example, in Paulsen, H. Angew. Chem. Int. Ed. (a) 1982, 21, 155; (b) 1990, 29, 823 and Schmidt, R. R. Angew. Chem. Int. Ed. 1986, 25, 212. These solution methodologies of oligosaccharide synthesis made dramatic advancements during the past few years described, for example, in (a) Fugedi, P.; Garegg, P. J.; Lonn, H.; Norberg, T. Glycoconjugate J. 1987, 4, 97; (b) Mootoo, D. R.; Date, V.; Fraser-Reid, B. J. Am. Chem. Soc. 1988, 110, 2662; (c) Veeneman, G. H.; Van Leeuwen, S. H.; Zuurmond, H.; Van Boom, J. H. J. Carbohydr. Chem. 1990, 6, 783; (d) Kanie, O.; Kiso, M., Hasegawa, A. J. Carbohydr. Chem. 1988, 7, 501; (e) Reddy, G. V.; Mereyala, H. B. Tetrahedron Lett. 1991, 47, 6435; (f) Friesen, R. W.; Danishefsky, S. J. J. Am. Chem. Soc. 1989, 111, 6656; (g) Friesen, R. W.; Danishefsky, S. J. Tetrahedron 1990, 46, 103. Still yields in the key glycosidic linkage formation steps are in the 80% range at best. In addition, certain "difficult linkages" are accessible in much lower yield, often below 50%. This reflects both the low reactivity and the instability of the reactants, in particular of the glycosylating agent. The activated glycosylating agent may decompose to several products, behaving chromatographically similar to the desired product. The excess glycosylating agent necessary to obtain an acceptable yield of coupled products often leads to reaction mixtures in which the desired compound is a relatively minor component. Thus a major obstruction to greater efficiency of glycosylation is the need for the chromatographic purification. In addition, each glycosidic linkage can form two stereoisomers (anomers) and this anomericity must be controlled. The control of anomeric specificity of glycosylation reactions performed in solutions was established in certain situations through the use of participating groups.
Methods employing enzymes for synthesis of oligosaccharides have been disclosed as well. The enzymes are either glycosyl transferases or glycosidases that normally function in the biosynthesis of oligosaccharides in living cells. The art of using enzymes for the in vitro synthesis of oligosaccharides has been described in many publications, for instance (a) Kaur, K. J; Alton, G., Hindsgaul, O., Carbohydrate. Res. 1991, 210, 145; (b) Wong, C. H., Ichikawa, Y., Krach, T., et al., J. Amer Chem. Soc. 1991, 113, 8137. Major obstacles in using the enzyme methodology are the difficulties in obtaining the pure enzymes in sufficient quantities and in purification of the final product.
Solid-state synthesis of oligosaccharides had been described in publications and reviews by (a) Frechet, J. M. J.; Schuerch, C. J. Am. Chem. Soc. 1971, 93, 492. (b) Frechet, J. M. J.; Schuerch, C. Carbohydr. Res. 1972, 22, 399; (c) Mathur, N. K.; Narang, C. K.; Williams, R. E. Polymers as Aids in Organic Chemistry; Academic Press: New York, 1980; Chapter 6; (d) Frechet J. M. J. in Polymer-Supported Reactions in Organic Synthesis (Hodge, P.; Sherrington, D. C.; Eds.); Wiley, Chichester 1980, p. 293 & p. 407; (e) Zehavi, U. Advances in Carbohydr. Chem. Biochem. 1988, 46, 179; (f) Frechet, J. M. J. Tetrahedron 1981, 37, 663. Among the problems encountered in using this methodology were: decreased glycosylation reaction rates compared to solution strategies, incomplete coupling, and lack of complete stereoselectivity. However, since two epimers (anomers) can be formed, stereochemical control is mandatory for successful synthesis of any oligosaccharide. This methodology has been considered as unsuitable for oligosaccharide synthesis because anomeric specificity could not be controlled in this reaction arrangement and the yields were low.
Polyethyleneglycol monomethylether (PEG) has been used as support for the synthesis of oligomers of peptides and nucleotides in polymer-assisted liquid synthesis as described for instance in (a) Bonora, G. M.; Scremin C. L.; Colonna, F. P.; Garbesi, A. Nucl. Acids Res. 1990, 18, 3155; (b) Kamaike, K; Hasegawa, Y.; Ishido, Y. Tetrahedron Lett. 1988, 29, 647; (c) Bayer, E.; Mutter, M. Nature 1972, 237, 512; (d) Bayer, E.; Mutter, M. The Peptides (Gross, E.; Meienhofer, J.; Eds.); Academic Press: New York 1980, 2, 286. In this reaction design the reactants are soluble in the reaction medium during the reaction itself. This methodology has not been utilized in oligosaccharide synthesis since it was considered a branch of solid-state design which has been shown to be unsuitable for the synthesis of oligosaccharides.